This invention relates to a method of ultrafiltration. More specifically, the invention relates to a method for purifying sequencing reactions.
Ultrafiltration with small devices is becoming a standard procedure used in DNA and protein research which is steadily requiring smaller and smaller quantities of materials. Methods of ultrafiltration have, in the past, relied on centrifugal forces to filter a liquid component through an ultrafiltration membrane. However, as the quantities of materials needed have become increasingly smaller, centrifugal methods no longer suit current needs, especially because these centrifugal methods are not conducive to automation.
Specifically, for example, DNA sequencing reactions must be purified prior to analysis by automated fluorescent sequencing (AFS) conducted on sequencing instruments such as the models ABI 377 and ABI 3700 from PE Biosystems, MegaBACE 1000 from Amersham Pharmacia Biotech and CEQ 2000 from Beckman. Introduction of the latter three high throughput capillary electrophoresis instruments has put new demands on sample purity and handling requirements.
Purification is required because contaminants that interfere with resolution of the sequencing products during electrophoretic separation will prevent determination of all or some of the DNA sequence. The identity of these interfering contaminants is determined, in part, by the sequencing chemistry used for labeling the sequencing products for fluorescent detection (e.g., dye primer or dye terminator chemistry), and the type of DNA sequencing instrument used for electrophoretic resolution of the labeled sequencing products (e.g., slab polyacrylamide gel or capillary electrophoresis).
Dye terminators are fluorescently labeled dideoxynucleotide triphosphates (ddNTP""s) which, once incorporated in a base-specific manner, prevent further polymerization of the sequencing product. Primers, template, DNA polymerase, dNTP""s, buffer, salts and dye terminators are added to a sequencing reaction at concentrations sufficient to allow production of sequencing products ranging from approximately 10 to 1200 nucleotides in length. Because dye terminators are not natural substrates of DNA polymerase, high concentrations must be provided relative to the natural dNTP substrates to ensure their incorporation into the polymerizing sequencing products. The consequence of this inefficient incorporation is that a large amount of unincorporated dye terminator is still present after the reaction is completed. Unincorporated dye terminators co-migrate with short sequencing products during electrophoresis, and produce a variety of artifacts that interfere with sequence analysis.
The most common methods for removing unincorporated dye terminators from sequencing reactions prior to electrophoresis are alcohol precipitation, typically using ethanol, and gel filtration. However, salts compete with sequencing products for electrokinetic injection onto capillary sequencing instruments and must also be removed. Ethanol precipitation has poor salt removal capabilities which detracts from its utility as a method for preparing samples prior to capillary electrophoresis because the efficiency of electrokinetic injection of sequencing products is inversely proportional to the salt concentration. As such, for purposes of removing these salts, alcohol precipitation is a poor and variable method for preparing samples. Although gel filtration is better suited for removing salt than alcohol precipitation, both gel filtration and alcohol precipitation are centrifuge-based methods which, as noted, are difficult to automate. Centrifugal methods are often sufficient for low throughput DNA sequencing carried out on older slab gel sequencing instruments, but the Genomics industry is scaling up DNA sequencing to a point where centrifugal sample preparation methods are no longer practical or sufficiently robust. Fueled by fierce competition and by new high throughput capillary DNA sequencing instruments, the Genomics industry is demanding unprecedented sample purity, automation capability and high throughput. However, such automatable methods, which are capable of removing salts and dye terminators, are not currently available.
Similarly, purified primer extension products may also be analyzed by gel analysis, such as capillary electrophoresis, or by mass spectrometry. For example, single nucleotide polymorphisms (SNPs) are single-base differences that genetically distinguish individuals within a population and as such can act as markers for diseases caused by the interaction of multiple genes. Differential termination of primer extension reactions is a commonly used method for detecting SNPs, whereby a dideoxy nucleotide analog is incorporated at the site of the sequence variation. Primer extension reactions contain many of the same components as sequencing reactions used to generate dideoxy-terminated sequencing ladders of indeterminant length. As such, the same contaminants must likewise be removed prior to analysis. The contaminants may comprise salts and dideoxy terminators, both of which pass through an ultrafiltration membrane, while the primer extension products do not.
It is therefore a primary object of this invention to provide a method for purifying DNA sequencing reactions or SNP assays that is capable of automation.
It is a further object of this invention to provide an automated method of ultrafiltration capable of removing salts and dye terminators.
It is a further object of this invention to provide a method of ultrafiltration capable of producing greater sample purity and recovery.
It is a further object of this invention to provide a method of ultrafiltration capable of high throughput.
It is a further object of this invention to provide an automated method for removing salts and dye terminators from DNA sequencing reactions capable of high throughput.
It is a further object of this invention to provide a method for removing salts from DNA sequencing reactions which is more efficient and consistent than salt removal by alcohol precipitation.
The method of the invention is the result of efforts to develop an ultrafiltration-based sequencing reaction cleanup method that is capable of automation and high throughput. Separation of impurities, such as salts and dye terminators, can be driven by vacuum using common laboratory equipment and, as such, is amenable to automation. Historically, ultrafiltration membranes in centrifugal devices were used to remove low molecular weight contaminants from higher molecular weight solutes, but fractionation can be time-consuming and inefficient. As noted, DNA sequencing reaction cleanup is a demanding separation wherein unincorporated fluorescent labels and salts must be removed from polymerase extension products in order to produce usable data. Unlike centrifugal ultrafiltration, which cannot produce sufficient purity, constant pressure differential ultrafiltration is used in the method of the invention to accomplish this critical separation. Constant pressure differential ultrafiltration provides highly pure results when the membrane and operating conditions are carefully controlled. An ultrafiltration membrane is employed to retain sequencing products while unincorporated fluorescent labels and salts pass through the membrane during constant pressure differential ultrafiltration.
As noted, constant pressure differential ultrafiltration offers excellent salt removal, fluorescent label removal and sequencing product recovery in a readily automated format. Vacuum-based or positive pressure-based manifolds used in the method are easier to automate than centrifugal ultrafiltration. Also, complete top access (no filtrate collection capabilities necessary) to the purified sequencing reaction allows direct electrokinetic injection from the surface of the ultrafiltration membrane, thereby simplifying sample processing and reducing the need for other consumables. Development of this novel protocol for processing sequencing reactions also eliminates artifacts which interfere with analyzing DNA sequence data.
A preferred method of the invention for sequencing reaction cleanup adapted to remove contaminants from a sequencing reaction, comprises the steps of: providing a defined quantity of sequencing reaction product; providing at least one ultrafiltration membrane having at least one surface; transferring the sequencing reaction product to the surface of the ultrafiltration membrane; and applying a first constant pressure differential to the ultrafiltration membrane at a force capable of producing the sequencing reaction product substantially free of the contaminants. For purposes of the method, the term xe2x80x9cproductxe2x80x9d may comprise samples of varying purity from crude samples, to samples that are partially purified and may comprise liquids and/or solids. Also, the term xe2x80x9csequencing reactionxe2x80x9d may comprise any polymerase extension reaction that produces a nucleic acid terminated in a dideoxynucleotide or modified dideoxynucleotide. For example, the term xe2x80x9csequencing reactionxe2x80x9d may comprise primer extension reactions. The method of the invention may be employed at any point during the process for concentrating and purifying sequencing reaction product, e.g. at the beginning with a crude sample which includes template or towards the end of the purification process as a final purification step. The ultrafiltration membrane may have a molecular weight cutoff between about 1000 and 30,000 Daltons and preferably has a molecular weight cutoff between about 3,000 and 15,000 Daltons. When the contaminants to be removed comprise salt and/or dye terminators, the step of applying a first constant pressure differential to the ultrafiltration membrane preferably comprises applying a force capable of producing the sequencing reaction product substantially free of salts and/or dye terminators.
The method may further comprise the step of suspending the quantity of sequencing reaction product in a defined volume of a first solvent. The step of suspending the quantity of sequencing reaction product in a defined volume of a solvent may comprise adding one or more solvents including, but not limited to, 10-15% formamide, xe2x89xa60.5 mM EDTA, and Milli-Q water to the sequencing reaction. Further, the step of applying a constant pressure differential to the ultrafiltration membrane may comprise applying vacuum at between about 5 to 28 inches of mercury, wherein the method may further comprise the steps of: washing the sequencing reaction product substantially free of the contaminants with a second solvent; and applying a second constant pressure differential to the ultrafiltration membrane, wherein the second constant pressure differential may also be applied at a force of between about 5 to 28 inches of mercury. The second solvent preferably comprises formamide, distilled water and/or EDTA.
The method may also further comprise the steps of: washing the sequencing reaction product substantially free of the contaminants with a third solvent; and applying a third constant pressure differential to the ultrafiltration membrane, wherein the third solvent may comprise Milli-Q water, formamide or EDTA. The third constant pressure differential vacuum is preferably applied at a force of between about 5 to 28 inches of mercury.
The method of the invention may further comprise the step of recovering the sequencing reaction product substantially free of the contaminants by pipetting off the surface of the membrane. In addition, the ultrafiltration membrane generally has an upstream and a downstream surface, wherein the suspended sequencing reaction product is transferred to and recovered from the upstream surface. The sequencing reaction product, substantially free of the contaminants, may be recovered from the retentate surface of the membrane by several methods including, but not limited to, pipetting, diffusion, agitation and electrokinetic transport. A vacuum force of about 5 inches of mercury may be applied during the recovery step to prevent any back migration of contaminants from the membrane substructure back to the retained material.
Depending on whether the product is to be analyzed by mass spectrometry or electrophoresis, the method may also comprise one or more of the following steps: washing said sequencing product with one or more salts capable of displacing any sodium ions from said product; resuspending said sequencing product, substantially free of one or more of said contaminants, in a matrix capable of crystallization; resuspending said sequencing product, substantially free of one or more of said contaminants, in water, EDTA or formamide for analysis by capillary electrophoresis; or resuspending said sequencing product, substantially free of one or more of said contaminants, in one or more volatile solvents for analysis by mass spectrometry.
As noted, the sequencing reaction product may comprise any polymerase extension reaction that produces a nucleic acid terminated in a dideoxynucleotide or modified dideoxynucleotide, including, but not limited to, one or more single nucleotide polymorphisms.
Another preferred method of the invention, for sequencing reaction cleanup adapted to remove contaminants from a sequencing reaction, comprises the steps of: providing a quantity of sequencing reaction product; suspending said quantity of sequencing reaction product in one or more solvents comprising formamide, EDTA and/or Milli-Q water; providing at least one ultrafiltration membrane having at least one surface; transferring said suspended sequencing reaction product to said surface of said ultrafiltration membrane; and applying a first constant pressure differential to said ultrafiltration membrane at a force between about 5 to 28 inches of mercury to produce said sequencing reaction product substantially free of said contaminants.
The method may further comprise the steps of: washing said sequencing reaction product substantially free of said contaminants with a second solvent; and applying a second constant pressure differential to said ultrafiltration membrane at a force between about 5 to 28 inches of mercury; wherein said second solvent preferably comprises formamide, EDTA and/or Milli-Q water. Still further, the method may comprise the steps of: washing said sequencing reaction product substantially free of said contaminants with a third solvent; and applying a third constant pressure differential to said ultrafiltration membrane; wherein said contaminants may comprise one or more salts and one or more dye terminators and wherein said step of applying a first constant pressure differential to said ultrafiltration membrane comprises applying a force capable of producing said sequencing reaction product substantially free of said salts and said dye terminators.
After the sequencing reaction product is substantially cleaned of the contaminants, the product may be resuspended in a matrix capable of crystallization, in water, formamide, EDTA or in one or more volatile solvents.